1. Field of the Invention
This invention relates to methods for performing an assay for determining an analyte by use of a conjugate of a member of a specific binding pair consisting of ligands and receptors, for example, antigens and antibodies, with a particle. The method of the invention has particular application to heterogeneous immunoassays of biological fluids, for example, serum or urine.
Luminescent compounds, such as fluorescent compounds and chemiluminescent compounds, find wide application because of their ability to emit light. For this reason, luminescers have been utilized as labels in assays such as immunoassays. For example, a member of a specific binding pair is conjugated to a luminescer and various protocols are employed. For example, the luminescer conjugate can be partitioned between a solid phase and a liquid phase in relation to the amount of analyte in a sample suspected of containing the analyte. By measuring the luminescence of either of the phases, one can relate the level of luminescence observed to a concentration of the analyte in the sample.
Particles, such as liposomes and erythrocyte ghosts, have been utilized as carriers of encapsulated water soluble materials. For example, liposomes have been employed to encapsulate biologically active material for a variety of uses, such as drug delivery systems wherein a medicament is entrapped during liposome preparation and then adminstered to the patient to be treated.
Particles, such as latex beads and liposomes, have also been utilized in assays. For example, in homogeneous assays an enzyme may be entrapped in the aqueous phase of a liposome labelled with an antibody or antigen. The particles are caused to release the enzyme in the presence of a sample and complement. Antibody or antigen-labelled particles, such as liposomes, having water soluble fluorescent or non-fluorescent dyes encapsulated within an aqueous phase of the lipid vesicle, have also been utilized to assay for analytes capable of entering into an immunochemical reaction with the surface bound antibody or antigen. Detergents have been used to release the dyes from the aqueous phase of the liposomes.
In cases in which the dye is incorporated within the aqueous layer of the particle, vesicle leakage is a problem. Fluorescent dyes incorporated in the aqueous layer have been observed to spill out as a result of some secondary effect of antigen-antibody binding. In addition, homogeneous methods involve no separation, and therefore, are subject to interferences from the sample.
It is, therefore, desirable to develop a new heterogeneous assay method, which has the ease and convenience of a homogeneous method, for determining an analyte in a sample. Additionally, it is also desirable to provide a sensitive heterogeneous immunoassay which provide amplification of the signal by releasing many detectable molecules per binding event without utilizing enzyme labels which require temperature and time controlled reactions. Direct labelling with luminescers, radio labels and the like, do not provide such an immunoassay. Such a sensitive immunoassay method, which is more fully described below, utilizes a conjugate of a specific binding pair member with a particle bearing a multiplicity of luminescer molecules. The luminescer is reversibly associated with a nonaqueous layer of the particle and can be released of the dye into solution during the assay to provide more sensitive determinations of the amount of analyte in the sample.
2. Description of the Related Art
O'Connell, et al., Clin. Chem., (1985) 31(9), 1424-1426 discloses a colorimetric immunoassay for digoxin utilizing large, unilamellar phospholipid vesicles having dye entrapped in the aqueous phase of the liposome. U.S. Pat. Nos. 3,850,578; 4,483,921; and 4,483,929 disclose immunoreactive liposome reagents in which antigen or antibody is bound to the surface of lipid vesicles. U.S. Pat. Nos. 4,529,561; 4,522,803; and 4,485,054 disclose a variety of methods for preparing lipid vesicles. U.S. Pat. No. 4,311,712 discloses a process for preparing a freeze dried liposome mixture. U.S. Pat. No. 4,588,578 discloses a method for the preparation of monophasic lipid vesicles and the use of such vesicles for drug delivery systems. U.S. Pat. No. 4,576,912 discloses a method of enhancing the fluorescent level of an immunoassay using certain long-chain carriers tagged with a plurality of fluorophores.